Objective:

Through this experiment you will able to understand the preparation of equilibrated phenol and its role in the biological or molecular experiments.

Theory:

Phenol, also known as carbolic acid, is an organic compound with the chemical formula C6H5OH. It is a white, crystalline solid. It has to be handled with care because it causes white blistering to the skin immediately. The crystals are often rather wet and discolored. Phenol contains a phenyl group which is bonded to a hydroxyl (-OH) group. Phenol used in DNA/RNA extraction to removes non-polar proteins, lipid residues etc.

Most batches of commercial liquefied phenol are clear and colourless and can be used in molecular cloning without re-distillation. Occasionally, batches of liquefied phenol are pink or yellow, and these should be rejected and returned to the manufacturer. Crystalline phenol is not recommended because it must be redistilled at 160°C to remove oxidation products, such as quinones, that cause the breakdown of phosphodiester bonds or cause cross-linking of RNA and DNA. Before use, phenol must be equilibrated to a pH of >7.8 because the DNA partitions into the organic phase at acid pH.

Phenol readily undergoes oxidation and must therefore be redistilled before use. Since redistilled phenol is acidic, a neutralization step with Tris base and then an equilibration step with TE (pH 8.0) or a similar buffer must be performed. Equilibrated phenol is stored at -20 °C.